Posttranslational modification by the small ubiquitin-related modifier (SUMO) is one of the crucial cellular processes in Giardia lamblia, a protozoan pathogen. In this study, 5 candidate SUMO substrate proteins of G. lamblia trophozoites were chosen based on their enrichment through affinity chromatography using a SUMO-interaction motif: never in mitosis A-related kinase (NEK), aminoacyl-histidine dipeptidase (AHD), protein disulfide isomerase 2 (PDI2), alcohol dehydrogenase 3, and ornithine carbamoyltransferase. Transgenic Giardia trophozoites expressing 1 of the 5 candidate SUMO substrate proteins were constructed, and their expression was confirmed by western blot using hemagglutinin-tag. Arginine deiminase (ADI) protein was expressed in Giardia trophozoites as a positive control. Cell extracts were processed for affinity chromatography using SUMO-interaction motif resin. As expected, the SUMOylated form of ADI was detected in the affinity chromatography extracts of ADI-expressing cells. Among the 5 candidate proteins, SUMOylated forms of NEK, AHD, and PDI2 were identified in the affinity chromatography extracts. These results suggest that NEK, AHD, and PDI2 activity is modulated via SUMOylation in Giardia trophozoites.

Intraflagellar transport (IFT) particles, a multi-protein apparatus composed of complex A and B, are known to be involved in homeostasis of flagella formation. IFT particles have recently become an interesting topic in Giardia lamblia, which has 4 pairs of flagella. In this experiment, we examined the function of giardial IFT components. When 7 components (IFT121, 140, 20, 46, 52, 81, and 88) of IFT were expressed in Giardia trophozoites as a tagged form with mNeonGreen, all of them were found in both flagella pores and cytoplasmic axonemes. In addition, motor proteins for IFT particles (kinesin-13 and kinesin-2b), were localized to a median body and cytoplasmic flagella, respectively. The CRISPRi-mediated knockdown of IFT88 significantly affected the lengths of all 4 flagella compared to the control cells, Giardia expressing dead Cas9 using control guide RNA. Decreased expression of kinesin-2b also resulted in shortening of flagella, excluding the ventral flagella. Live Giardia cells expressing IFT88-mNeonGreen clearly demonstrated fluorescence in flagella pores and cytoplasmic axonemes. These results on IFT88 and kinesin-2b indicate that IFT complex plays a role in maintenance of G. lamblia flagella.